To investigate how a shallow dose-response curve might arise (as seen in data analyzed in Fallahi-Sichani et al. (2013) Nature Chemical Biology. PMID: 24013279), this assay focuses on drugs inhibiting the PI3K/Akt/mTOR pathway that varied widely in Hill slope (HS) and Emax values independent of proliferation rate. For six compounds with varying HS, target inhibition was measured by immunofluorescence microscopy and cell killing in four breast cell lines (HER2-amplified AU565 and HCC1954 cancer cells, hormone receptor-positive T47D cancer cells, and non-transformed MCF10A cells). The effects of the mTOR inhibitor PP242, PI3K inhibitor GSK1059615, dual specificity mTOR/PI3K inhibitor dactolisib (BEZ235), Akt inhibitors MK2206 and triciribine, and also an EGFR inhibitor gefitinib were probed 6 hr and 24 hr after drug exposure in 9-point dose-response assays using antibodies specific for p-ERK (Thr202/Tyr204), p-Akt (Ser473), p-4EBP1 (Thr37/46), and p-S6 (Ser235/236). A shallow dose-response curve is correlated with high cell-to-cell variability in target (p-4EBP1) inhibition by PP242 and dactolisib as compared to drugs for which HS ~ 1 or HS > 1 (in four of four cell lines tested). This dataset refers to data shown in Figure 5 and Supplementary Figures 5, 7, 8 and 9 of the paper.

Assay Protocol:

AU565 and HCC1954 cells were cultured in RPMI 1640 (ATCC) supplemented with 10% FBS (FBS). T47D cells were cultured in RPMI 1640 supplemented with 10% FBS and insulin (0.2 U/ml). MCF10A cells were cultured in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, EGF (20 ng/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml) and cholera toxin (100 ng/ml). Penicillin (50 U/ml) and streptomycin (50 μg/ml) were added to all growth media. All of the compounds were dissolved in DMSO as 10-mM stock solutions. For dose-response experiments, cells were plated in two repli­cates at 7,000 cells per well in 96-well plates (Corning) in full growth medium for 24 h and then treated them with nine doses in serial dilutions (100 pM to 10 uM) of each compound for 6 h and 24 h.